Objective To investigate the effects of titanium modified by ultrasonic acid etching/anodic oxidation (UAT) loaded with endothelial progenitor cells-exosome (EPCs-exo) on proliferation and osteogenic and angiogenic differentiations of adipose-derived stem cells (ADSCs). Methods The adipose tissue and bone marrow of 10 Sprague Dawley rats were harvested. Then the ADSCs and EPCs were isolated and cultured by collagenase digestion method and density gradient centrifugation method, respectively, and identified by flow cytometry. Exo was extracted from the 3rd to 5th generation EPCs using extraction kit, and CD9 and CD81 were detected by Western blot for identification. The three-dimensional printed titanium was modified by ultrasonic acid etching and anodic oxidation to prepare the UAT. The surface characteristics of UAT before and after modification was observed by scanning electron microscopy; UAT was placed in EPCs-exo solutions of different concentrations (100, 200 ng/mL), and the in vitro absorption and release capacity of EPCs-exo was detected by BCA method. Then, UAT was placed in DMEM medium containing different concentrations of EPCs-exo (0, 100, 200 ng/mL), and co-cultured with the 3rd generation ADSCs to construct UAT-ADSCs-exo. Cell morphology by laser confocal microscopy, live/dead cell staining, and cell proliferation were observed to evaluate biocompatibility; alkaline phosphatase (ALP) staining and alizarin red staining, RT-PCR detection of osteogenesis-related genes [osteocalcin (OCN), RUNT-related transcription factor 2 (Runx2), ALP, collagen type 1 (COL-1)] and angiogenesis-related gene [vascular endothelial growth factor (VEGF)], immunofluorescence staining for osteogenesis (OCN)- and angiogenesis (VEGF)-related protein expression were detected to evaluate the effect on the osteogenic and angiogenic differentiation ability of ADSCs. Results Scanning electron microscopy showed that micro-nano multilevel composite structures were formed on the surface of UAT. About 77% EPCs-exo was absorbed by UAT within 48 hours, while EPCs-exo absorbed on the surface of UAT showed continuous and stable release within 8 days. The absorption and release amount of 200 ng/mL group were significantly higher than those of 100 ng/mL group (P<0.05). Biocompatibility test showed that the cells in all concentration groups grew well after culture, and the 200 ng/mL group was better than the other groups, with fully spread cells and abundant pseudopodia, and the cell count and cell activity were significantly higher than those in the other groups (P<0.05). Compared with the other groups, 200 ng/mL group showed enhanced ALP activity and mineralization ability, increased expressions of osteogenic and angiogenic genes (OCN, Runx2, COL-1, ALP, and VEGF), as well as increased expressions of OCN and VEGF proteins, with significant differences (P<0.05). Conclusion EPCs-exo can effectively promote the adhesion, proliferation, and osteogenic and angiogenic differentiation of ADSCs on UAT surface, the effect is the most significant when the concentration is 200 ng/mL.
ObjectiveTo summarize the research progress of the effects of high glucose microenvironment on the biological activity of adipose-derived stem cells (ADSCs).MethodsThe literature on the high glucose microenvironment and ADSCs at home and abroad in recent years was reviewed, and the effects of high glucose microenvironment on the general characteristics, differentiation potential, angiogenesis, and nerve regeneration of ADSCs were summarized.ResultsThe accumulation of advanced glycosylation end products (AGEs) in the high glucose microenvironment led to changes in the biological activities of ADSCs through various pathways, including cell surface markers, proliferation, migration, multi-lineage differentiation, secretory function, and tissue repair ability. The ability of ADSCs to promote angiogenesis and nerve regeneration in high glucose microenvironment is still controversial.ConclusionHigh glucose microenvironment can affect the biological activity of ADSCs, and the effect and mechanism of ADSCs on angiogenesis and nerve regeneration in high glucose microenvironment need to be further studied.
ObjectiveTo investigate the effect of human subcutaneous adipose-derived stem cells (hADSCs) local transplantation on orthodontically induced root resorption (OIRR) and provide theoretical and experimental basis for the clinical application of hADSCs to inhibit OIRR. MethodsForty 8-week-old male Sprague Dawley rats were randomly divided into experimental group and control group, with 20 rats in each group, to establish the first molar mesial orthodontic tooth movement (OTM) model of rat right maxillary. The rats in the experimental group were injected with 25 μL of cell suspension containing 2.5×105 hADSCs on the 1st, 4th, 8th, and 12th day of modeling, while the rats in the control group were injected with 25 μL of PBS. The rat maxillary models were obtained before and after 7 and 14 days of force application, and 10 rats in each group were killed and sampled after 7 and 14 days of force application. The OTM distance was measured by stereomicroscope, the root morphology of the pressure side was observed by scanning electron microscope and the root resorption area ratio was measured. The root resorption and periodontal tissue remodeling of the pressure side were observed by HE staining and the root resorption index was calculated. The number of cementoclast and osteoclast in the periodontal tissue on the pressure side was counted by tartrate resistant acid phosphatase staining. Results The TOM distance of both groups increased with the extension of the force application time, and there was no significant difference (P<0.05). There was no significant difference in OTM distance between the experimental group and the control group after 7 and 14 days of force application (P>0.05). Scanning electron microscope observation showed that small and shallow scattered resorption lacunae were observed on the root surface of the experimental group and the control group after 7 days of force application, and there was no significant difference in the root resorption area ratio between the two groups (P>0.05); after 14 days of application, the root resorption lacunae deepened and became larger in both groups, and the root resorption area ratio in the experimental group was significantly lower than that in the control group (P<0.05). The range and depth of root absorption in the experimental group were smaller and shallower than those in the control group, and the root absorption index in the experimental group was significantly lower than that in the control group after 14 days of force application (P<0.05). The number of cementoclast in the experimental group was significantly lower than that in the control group after 7 and 14 days of force application (P<0.05); the number of osteoclasts in the experimental group was significantly lower than that in the control group after 14 days of force application (P<0.05). Conclusion Local transplantation of hADSCs may reduce the area and depth of root resorption by reducing the number of cementoclasts and osteoclasts during OTM in rats, thereby inhibiting orthodontic-derived root resorption.
Objective To investigate the effects of adipose-derived stem cells (ADSCs) and endothelial cells (ECs) on the survival and neovascularization of fat tissue transplants. Methods The ADSCs were isolated by collagenase digestion from the adipose tissues voluntarily donated by the patients undergoing mastectomy, and subcultured. The passage 3 ADSCs were used for subsequent experiments. The residual fat tissues were used to prepare fat particles (FPs). The human umbilical vein endothelial cells (HUVECs) were used as ECs for subsequent experiments. Eighty healthy male nude mice, aged 4-6 weeks, were randomly divided into 4 groups (n=20). The mice were received subcutaneous injection at the dorsum of 1 mL FPs+0.3 mL normal saline (NS) in control group, 1 mL FPs+2×106 ECs+0.3 mL NS in ECs group, 1 mL FPs+2×106 ADSCs+0.3 mL NS in ADSCs group, and 1 mL FPs+1×106 ECs+1×106 ADSCs+0.3 NS in ADSCs+ECs group. General observations of the injection sites were performed, and the survival of the mice was recorded. At 2, 4, 8, and 12 weeks after injection, grafted fat tissues were firstly assessed by ultrasonography, then they were collected for volume measurement (water displacement method) and histology observation (HE staining and immunofluorescence staining). Results All mice survived until the end of experiment. At each time point, no significant difference was noted between groups in ultrasonography assay. There was no significant blood flow signal in the grafted fat tissues, or cysts, calcification, solid occupying in recipient area. Generally, the volume of grafted fat tissues decreased with time in all groups. Specifically, the volumes of grafted fat tissues were larger in ADSCs group and ADSCs+ECs group than that in control group and ECs group (P<0.05) at each time point, and in ADSCs group than in ADSCs+ECs group (P<0.05) at 8 and 12 weeks. HE staining showed that all groups had similar tendencies in general histology changes, and remodeling in ADSCs group was the fastest than in the other groups. By immunofluorescence staining for neovascularization, the new vessels in all groups were increasing with time. The vessel densities were higher in ECs group, ADSCs group, and ADSCs+ECs group than in control group (P<0.05) at each time point, in ADSCs group than in ECs group and ADSCs+ECs group (P<0.05) at 4 weeks, in ADSCs group and ADSCs+ECs group than in ECs group (P<0.05) at 8 and 12 weeks. Conclusion ADSCs can significantly increase the survival of transplanted fat tissue, which may be related to promoting the neovascularization.
ObjectiveTo explore the feasibility of three-dimensional (3D) bioprinted adipose-derived stem cells (ADSCs) combined with gelatin methacryloyl (GelMA) to construct tissue engineered cartilage.MethodsAdipose tissue voluntarily donated by liposuction patients was collected to isolate and culture human ADSCs (hADSCs). The third generation cells were mixed with GelMA hydrogel and photoinitiator to make biological ink. The hADSCs-GelMA composite scaffold was prepared by 3D bioprinting technology, and it was observed in general, and observed by scanning electron microscope after cultured for 1 day and chondrogenic induction culture for 14 days. After cultured for 1, 4, and 7 days, the composite scaffolds were taken for live/dead cell staining to observe cell survival rate; and cell counting kit 8 (CCK-8) method was used to detect cell proliferation. The composite scaffold samples cultured in cartilage induction for 14 days were taken as the experimental group, and the composite scaffolds cultured in complete medium for 14 days were used as the control group. Real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect cartilage formation. The relative expression levels of the mRNA of cartilage matrix gene [(aggrecan, ACAN)], chondrogenic regulatory factor (SOX9), cartilage-specific gene [collagen type Ⅱ A1 (COLⅡA1)], and cartilage hypertrophy marker gene [collagen type ⅩA1 (COLⅩA1)] were detected. The 3D bioprinted hADSCs-GelMA composite scaffold (experimental group) and the blank GelMA hydrogel scaffold without cells (control group) cultured for 14 days of chondrogenesis were implanted into the subcutaneous pockets of the back of nude mice respectively, and the materials were taken after 4 weeks, and gross observation, Safranin O staining, Alcian blue staining, and collagen type Ⅱ immunohistochemical staining were performed to observe the cartilage formation in the composite scaffold.ResultsMacroscope and scanning electron microscope observations showed that the hADSCs-GelMA composite scaffolds had a stable and regular structure. The cell viability could be maintained at 80%-90% at 1, 4, and 7 days after printing, and the differences between different time points were significant (P<0.05). The results of CCK-8 experiment showed that the cells in the scaffold showed continuous proliferation after printing. After 14 days of chondrogenic induction and culture on the composite scaffold, the expressions of ACAN, SOX9, and COLⅡA1 were significantly up-regulated (P<0.05), the expression of COLⅩA1 was significantly down-regulated (P<0.05). The scaffold was taken out at 4 weeks after implantation. The structure of the scaffold was complete and clear. Histological and immunohistochemical results showed that cartilage matrix and collagen type Ⅱ were deposited, and there was cartilage lacuna formation, which confirmed the formation of cartilage tissue.ConclusionThe 3D bioprinted hADSCs-GelMA composite scaffold has a stable 3D structure and high cell viability, and can be induced differentiation into cartilage tissue, which can be used to construct tissue engineered cartilage in vivo and in vitro.
Objective To investigate the possibility of enhancing the inducing rate of adipose-derived stem cells (ASCs) into epidermal cells in the medium containing all-trans retinoic acid (ATRA) by supplementing with HaCaT condition medium. Methods ASCs were isolated and identified by detecting the expression of CD34, CD45, CD73, CD90, and CD105 with flow cytometry and differentiating into adipose and osteoblast lineage in the induction medium. The air-liquid interface cell culture model was established with the Transwell Room. The induction medium A contained ATRA, epidermal growth factor (EGF), and keratinocyte growth factor (KGF), while the induction medium B contained ATRA, EGF, KGF, and HaCaT condition medium. Experiment was divided into three groups cultured for 12 days: induction medium A (group A), induction medium B (group B), basic medium (group C). The epidermal cell surface markers: cytokeratin (CK) 14, 15, 16, 19 (Pan-CK) were detected by flow cytometry and CK14 were identified by immunofluorescence stain. Results After induction for 12 days, flow cytometry showed that the positive rate of Pan-CK in group B [(22.0±3.5)%] was higher than that in group A [(11.9±2.7)%], which were both higher than that in group C [(1.1±0.3)%], and the differences were statistical significantly (P<0.01). Immunofluorescence stain showed that the positive rate of CK14 in group B was higher than that in group A [(19.5±7.0)%vs. (10.8±5.7)%, P<0.01], and the expression of CK14 was negative in group C. Conclusion HaCaT condition medium can enhance the ability of ASCs differentiation into epidermal cells in the culture medium containing ATRA.
ObjectiveTo investigate the microRNA (miRNA) expression profile during chondrogenic differentiation of human adipose-derived stem cells (hADSCs), and assess the roles of involved miRNAs during chondrogenesis. MethodshADSCs were harvested and cultured from donors who underwent elective liposuction or other abdominal surgery. When the cells were passaged to P3, chondrogenic induction medium was used for chondrogenic differentiation. The morphology of the cells was observed by inverted phase contrast microscopy. Alcian blue staining was carried out at 21 days after induction to access the chondrogenic status. The expressions of chondrogenic proteins were detected by ELISA at 0, 7, 14, and 21 days. The miRNA expression profiles at pre- and post-chondrogenic induction were obtained by microarray assay, and differentially expressed miRNAs were verified by real-time quantitative PCR (qRT-PCR). The targets of the miRNAs were predicted by online software programs. ResultshADSCs were cultured successfully and induced with chondrogenic medium. At 21 days after chondrogenic induction, the cells were stained positively for alcian blue staining. At 7, 14, and 21 days after chondrogenic induction, the levels of collogen type Ⅱ, Col2a1, aggrecan, Col10a1, and chondroitin sulfate in induced hADSCs were significantly higher than those in noninduced hADSCs (P<0.05). Eleven differentially expressed miRNAs were found, including seven up-regulated and four down-regulated. Predicted target genes of the differentially expressed miRNAs were based on the overlap from three public prediction algorithms, with the known functions of regulating chondrogenic differentiation of stem cells, selfrenewal, signal transduction, intracellular signaling cascade, and cell cycle control. ConclusionA group of miRNAs and their target genes are identified, which may play important roles in regulating chondrogenic differentiation of hADSCs. These results will facilitate the initial understanding of the molecular mechanism of chondrogenic differentiation in hADSCs and subsequently control hADSCs differentiation, and provide high performance seed cells for cartilage tissue engineering.
Objective To review the research progress of miRNA regulation in the differentiation of adipose-derived stem cells (ADSCs). Methods The recent literature associated with miRNAs and differentiation of ADSCs was reviewed. The regulatory mechanism was analyzed in detail and summarized. Results The results indicate that the expression of miRNAs changes during differentiation of ADSCs. In addition, miRNAs regulate the differentiation of ADSCs into adipocytes, osteoblasts, chondrocytes, neurons, and hepatocytes by regulating the signaling pathways involved in cell differentiation. Conclusion Through controlling the differentiation of ADSCs by miRNAs, the suitable seed cell for tissue engineering can be established. The review will provide a theoretical basis for molecular targeted therapy and stem cell therapy in clinic.
Objective To assess the effect of pregnant rat adipose-derived stem cells (ADSCs) on repair of acute liver injury. Methods ADSCs were isolated from 18-week pregnant Sprague Dawley rats and were identified by flow cytometry. Twenty Sprague Dawley rats were randomly divided into groups A, B, C, and D (n=5); rats in group A were not treated as normal controls; rats in groups B, C, and D were injected intraperitoneally with CCl4 to establish the acute liver injury model. At 2 hours after modeling, DPBS, 0.1 mL normal rat ADSCs (2×106cells/mL), and pregnant rat ADSCs (2×106cells/mL) were injected into the spleen in groups A, C, and D respectively; rats in group B was not treated. After 7 days, total bilirubin (TBIL), alanine aminotransferase (ALT), aspartic acid transaminase (AST), albumin (ALB), and total protein (TP) in serum were measured. The liver tissue sections were stained with HE. The expressions of Ki67, alpha-fetoprotein (AFP), and ALB were measured by immunohistochemistry. Results The serum levels of TBIL, ALT, and AST in group B were significantly higher than those in groups A, C, and D (P<0.05), but ALB and TP were significantly lower than those in groups A, C, and D (P<0.05). The levels of TBIL, ALT, and AST were significantly higher in groups C and D than group A, and in group C than group D (P<0.05). There was no significant difference in serum levels of ALB among groups A, C, and D (P>0.05). The serum level of TP in groups C and D was significantly lower than that in group A (P<0.05), but no significant difference was found between group C and group D (P>0.05). HE staining showed that the liver tissue of group A had clear structure; the cells arranged neatly with uniform size. The hepatocytes in group B showed obvious edema, disorderly arrangement, dot necrosis in liver lobules, and diffuse infiltration of inflammatory cells. In groups C and D, the inflammation and hepatocellular necrosis were obviously reduced when compared with group B, and the number of vacuoles caused by dilation of mitochondria and rough endoplasmic reticulum was decreased; especially in group D, improvement of liver injury was more effective. The Ki67 positive cell rate was significantly higher in groups C and D than groups A and B (P<0.05), in group B than group A (P<0.05), and in group D than group C (P<0.05). There was no expression of AFP in groups A and B, but positive expression was observed in groups C and D, and AFP positive cell rate of group D was significantly higher than that of group C (t=3.006,P=0.017). ALB expression was significantly higher in groups C and D than groups A and B (P<0.05), and in group D than group C (P<0.05). Conclusion Pregnant rat ADSCs could promote repair of liver injury induced by CCl4.
ObjectiveTo explore the effect of vascular endothelial growth factor 165 (VEGF165)-loaded porous poly (ε-caprolactone) (PCL) scaffolds on the osteogenic differentiation of adipose-derived stem cells (ADSCs).MethodsThe VEGF165-loaded porous PCL scaffolds (written, Sf-g/VEGF) were fabricated through a combination of solvent casting/salt leaching and a thermal-induced phase separation technique and then observed under scanning electron microscope (SEM). The release kinetics was determined by ELISA kit. The ADSCs were isolated from inguinal fat pads of 15 Sprague Dawley rats and cultured. The passage 3-4 ADSCs were seeded into the scaffolds, and then cultured in vitro for 7 days. The passage 3-4 ADSCs were seeded into the porous PCL scaffolds (written, Sf-g) as control. The alizarin red S (ARS) staining, ARS activity assay, and real-time quantitative PCR (RT-PCR) were performed to measure the osteogenic differentiation of ADSCs in vitro. Six Sprague Dawley rats were recruited to prepare the bilateral calvarial bone defects models (n=12). The 12 calvarial bone defects were randomly divided into 3 group (n=4). The defects of negative control group were not treated; the defects of Sf-g group and Sf-g/VEGF group were repaired with ADSCs-Sf-g scaffold complex and ADSCs-Sf-g scaffold complex, respectively. At 8 weeks after transplantation, the Micro-CT and HE staining were conducted to evaluate the osteogenic effects in vivo.ResultsThe morphology of the Sf-g/VEGF scaffolds were porous and well-connected, and the cumulative release rate was approximately 80% in 120 hours. The ARS staining showed that the ARS activity of Sf-g/VEGF group were stronger than that of Sf-g group (t=10.761, P=0.000). The mRNA expressions of osteogenic specific markers [special AT-rich sequence protein 2 (Satb2), alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN)] were significantly higher in Sf-g/VEGF group than in Sf-g group (P<0.05). The results of Micro-CT and HE staining also confirmed the promotion effect of Sf-g/VEGF scaffolds. All defects of 2 groups were partially repaired by new bone tissue, especially in Sf-g/VEGF group. The volume and area of new bone tissue were significantly higher in Sf-g/VEGF group than in Sf-g group (P<0.05).ConclusionThe VEGF165-loaded scaffolds can significantly improve the osteogenic differentiation of ADSCs both in vitro and in vivo.